Project Summary/Abstract Two protein-based vaccines are licensed in the U.S. for prevention of meningococcal group B (MenB) disease in adolescents and young adults. One vaccine contains two Factor H binding protein (FHbp) Ags, one from each of the two phylogenic sub-families; the other contains one FHbp sub-family B Ag and three other potentially protective Ags. The former vaccine has limitations in protection against strains with no or low FHbp expression (10-20% of strains) and the latter has limitations against the ~40% of strains with FHbp in sub-family A and lacking another matched Ag. Our previous studies in human complement Factor H (FH) transgenic mice and infant rhesus macaques showed that binding of FH to FHbp decreases the magnitude of the protective Ab responses. Further, our colleagues have shown that human FH decreases the protective Ab responses to an alternative FH ligand known as Neisserial surface protein A (NspA), which is present in virtually all MenB strains. Since recruitment of the complement down-regulator FH to the bacterial surface contributes to immune evasion, a vaccine that targets both of these FH ligands would elicit Abs that effectively defeat this virulence mechanism. In Aim 1, we will incorporate FHbp amino acid substitutions to decrease binding of FH into optimally cross-protective FHbp sequence variants in each sub-family. In Aim 2, we will introduce substitutions to decrease binding of human FH to NspA based on the known crystal structure of NspA and to increase solubility of the recombinant protein using random mutagenesis and a fluorescent reporter system. For both Aims 1 and 2, we will test the optimized antigens for FH and MAb binding in vitro, evaluate immunogenicity in wild-type and human FH transgenic mice and test the serum Ab protective activity. In Aim 3, we will develop a combination FHbp-NspA vaccine that will combine an engineered FHbp sequence variant from each sub-family and an engineered NspA Ag. We will test the combination vaccine compared to a licensed MenB vaccine, first in human FH transgenic mice then in infant rhesus macaques with high binding of FH to wild-type FHbp and NspA. For each of these studies, we will determine the magnitudes of the bactericidal Ab responses against MenB strains that are specific for each of the two Ags (i.e. lacking or with very low expression of one of the two Ags). We also will test passive Ab protection in human FH transgenic infant rats. The data will support development of a second-generation MenB vaccine that elicits higher and more sustained protective Ab responses and broader protection than the currently licensed vaccines. Since binding of complement regulators is a common microbial virulence mechanism, the results also will have broad implications for the development of vaccines against other microbes that bind complement regulators or other host molecules.